This invention relates to prenatal detection methods using non-invasive techniques and, more particularly, to the detection of fetal or maternal RNA in a blood sample from a pregnant subject, including the assessment of the gene expression pattern of an unborn fetus by analyzing the blood sample.
We have previously described the detection of circulating fetal DNA in maternal plasma and serum. Y. M. Dennis Lo et al., xe2x80x9cPresence of Fetal DNA in Maternal Plasma and Serum,xe2x80x9d Lancet 1997, 350:485-7. We have demonstrated the use of this technology for non-invasive prenatal diagnosis. Y. M. Dennis Lo et al., xe2x80x9cPrenatal Diagnosis of Fetal RhD Status by Molecular Analysis of Maternal Plasma,xe2x80x9d New England Journal of Medicine 1998, 339:1734-8. The discovery of fetal DNA in maternal plasma has opened up a new horizon on prenatal molecular diagnosis. A number of groups have since shown that fetal genetic traits, such as RhD status and inherited genetic diseases, can be determined from fetal DNA in maternal plasma.
The main limitation of this existing technology is that fetal DNA detection will only allow one to tell the presence and quantity of genetic material of fetal origin in the maternal circulation. It does not give information regarding the gene expression profile of the baby. Gene expression patterns can be expected to be affected by physiological or pathological processes affecting the baby and mother. Such direct monitoring of fetal gene expression is beyond the reach of conventional non-invasive technologies.
Embodiments of the present invention are directed to the detection of fetal or maternal RNA in a blood sample from a pregnant subject, and may involve subjecting the sample to a test for fetal or maternal analysis indicative of a fetal or maternal condition or characteristics. For instance, the RNA analysis may include the assessment of the gene expression pattern of an unborn fetus by analyzing a blood sample from the mother. The utility of the invention has been demonstrated from the second trimester and the third trimester of pregnancy, and may be used for the diagnosis and monitoring of a wide variety of diseases, including pre-eclampsia and preterm labor.
The new prenatal monitoring technology according to the present invention allows, for the first time, the detection of genes which are expressed by the fetus, just by analysis of a sample of maternal blood. This will become a platform technology upon which a new generation of non-invasive prenatal tests can be built.
The prenatal monitoring technology is based on the discovery of circulating RNA of fetal origin in the plasma of pregnant women. Heretofore, it was not known whether fetal RNA was also present in maternal plasma. Using a two-stepped reverse transcriptase polymerase chain reaction (RT-PCR) assay, we demonstrate the presence of fetal-derived, male-specific mRNA in plasma of pregnant women carrying male fetuses. As described herein, fetal RNA is detected by RT-PCR; but in principle any RNA detection method can be used.
This technology is expected to have application in all cases of pregnancy for monitoring the physiological or pathological status of a fetus. It is further anticipated that in certain scenarios, more than one such monitoring may be desirable or necessary during pregnancy. Thus, the number of potential clients and possibilities of multi-usage is potentially enormous.
In accordance with an aspect of the present invention, a method of performing prenatal monitoring or testing of a blood sample obtained from a pregnant subject comprises removing all or substantially all nucleated and a nucleated cell populations from the blood sample to obtain a remaining material. The remaining material is subjected to a test for fetal or maternal RNA analysis indicative of a fetal or maternal condition or characteristic. The remaining material may comprise plasma or serum.
In accordance with another aspect of the invention, a detection method performed on a maternal serum or plasma sample from a pregnant female comprises detecting the presence of RNA of fetal origin in the sample. The fetal RNA may be amplified to facilitate detecting the presence of RNA of fetal origin in the sample. For instance, the fetal RNA may be converted into complementary DNA by a reverse transcriptase and then detected by a polymerase chain reaction.
In some embodiments, the fetal RNA is detected using a sequence specific probe. The RNA detection may involve detecting the presence of a fetal RNA transcribed from the Y chromosome, or a fetal RNA from a gene or other DNA sequences inherited from either the father or the mother. The RNA may be detected by any one of a physical method, an immunological method, and a biochemical method.
In accordance with another aspect of the present invention, detection method performed on a maternal serum or plasma sample from a pregnant female comprises detecting the presence of RNA of fetal or maternal origin in the sample. The RNA detection may involve detecting the presence of RNA transcribed from genes on chromosome 6. The RNA may be transcribed from the HLA-G gene.
Another aspect of the invention is directed to a method of performing prenatal monitoring or testing. The method comprises providing a maternal blood sample, and separating the sample into a predominantly cellular fraction (fraction 1) and a predominantly non-cellular fraction (fraction 2). The presence of RNA of fetal origin is detected in fraction 2. The method further includes providing a diagnosis based on at least one of the following: presence, quantity, concentration, sequence, and biochemical composition of the detected RNA.
In accordance with another aspect of the present invention, a method of performing a prenatal monitoring or testing on a maternal blood sample comprises obtaining a non-cellular fraction of the maternal blood sample, and performing an RNA analysis for expressed human genes on the fraction.